We propose to study the mechanism of cell-cell interaction at two separate stages in the development of Dictyostelium: 1. During aggregation the cells respond to cAMP when it is bound to specific cAMP binding sites. Adenyl cyclase becomes activated for a brief period following binding of cAMP and then enters a refractory period. Concomitant with the activation/inactivation of adenyl cyclase, the level of methylation of membrane proteins increases upon addition of cAMP. We have built up considerable information on the complexity and changes in membrane proteins during aggregation and now can study their physiological roles. We plan to study the process of membrane protein methylation in response to cAMP and other cyclic nucleotides. We will characterize both the methylated products and the methylation mechanism. We will attempt to isolate and characterize adenyl cyclase in this system and study its activation/inactivation. We will characterize the cAMP binding proteins which we have solubilized from the membranes. The photo affinity label 8'azido 3'5' cAMP will be used to covalently label the binding proteins. The chemotactic response will then be analyzed in light of the biochemical properties of some of its components. 2. During the slug stage cells in the posterior undergo specific differentiations in preparation for transforming into spores while cells in the anterior fail to undergo these differentations. This position-determined differentiation is size invariant and regulates in response to surgical alterations. We will attempt to define the processes which result in pattern formation by the analysis of series of mutant strains in which the pattern is severely altered. The response of certain classes of mutants in chimeric mixtures with wild-type and other strains will limit the number of possible models to account for pattern formation. A detailed model based on diffusion of a specific effector will be critically evaluated in the light of the results. When the appropriate mutants are characterized, we will attempt to recognize and then purify the effector(s) directing the spatial pattern of differentiation.